PROJECT SUMMARY (Project 3 ? Optimal use of ART in establishing functional cures) In Project 3, Emmune, Inc. is collaborating with ViiV Healthcare to determine how antiretroviral therapy (ART) should be administered so as to maximize the ability of AAV-expressed therapeutics to establish robust functional cures and perhaps deplete the viral reservoir. ViiV Healthcare is developing long- acting cabotegravir, a product with several potential synergies with AAV-expressed anti-retroviral biologics developed by Emmune. This program seeks to ?functionally cure? HIV patients with a one-time intramuscular injection of an AAV-expressed therapeutic without the need for continuing antiretroviral therapy (ART). However, when AAV is first administered, ART is probably necessary to prevent the emergence of resistant viruses while the AAV-expressed therapeutic is present at suboptimal concentrations. Such resistance has been observed when broadly neutralizing antibodies (bNAbs) were administered to patients. The potential for resistance is an important reason why we have focused on eCD4-Ig, an immunoadhesin constructed from an antibody Fc and the parts of CD4 and CCR5. Because it closely emulates these HIV receptors, eCD4-Ig presents fundamental barriers to viral escape. Nonetheless, we have observed the emergence of partial resistance to eCD4-Ig, albeit associated with a high fitness cost. To prevent selection for partial resistance, we have pre-treated SHIV-infected macaques with ART before administering AAV expressing eCD4-Ig. When ART was subsequently withdrawn, transient ?blips? of viral RNA were occasionally detectable. We hypothesize that the quality of these functional cures can be improved by timing the application of ART so that host immune responses have not had time to wane prior introducing AAV expressing eCD4-Ig. Thus, AAV-expressed eCD4-Ig and peak host immune responses can work synergistically to deplete viral reservoirs and establish functional cures. In this project, we will determine whether ART is detrimental to depleting viral reservoirs and establishing functional cures in SHIV-infected macaques. To balance the need to prevent resistance with the benefits of synergy with host immune responses, we propose initiating ART and AAV- expressed eCD4-Ig at the same time. We also will determine whether a one-time administration of the long-acting formulation of cabotegravir, at the same time as AAV-eCD4-Ig is administered, is a practical solution for preventing partial resistance to eCD4-Ig. Conversely, we will also determine whether expression of eCD4-Ig prevents cabotegravir resistance during its so-called ?long tail?, when cabotegravir concentrations are sub-clinical. Finally, these efforts will be supported by a panel of immunological studies that will provide a mechanistic insight into how ART, eCD4-Ig and host immunity collaborate to establish robust functional cures.